The circadian clock of the liver controls intrinsic daily rhythms of gene expression that are essential for metabolic homeostasis. Germline deletion of the circadian clock in mice causes disorders of energy balance including hyperglycemia, hyperlipidemia, and obesity. Genes under direct control of the liver clock may influence the development of hepatic steatosis and provide timing cues for hepatocyte regeneration after liver injury. The mammalian clock is built upon a negative feedback loop that generates circadian rhythms at the molecular level. PERIOD (PER) proteins, acting in a large complex, enter the nucleus and repress the activity of CLOCK-BMAL1, the transcription factor driving their expression. The mechanism by which PER complexes cause transcriptional repression remains poorly defined. The goal of this application is to identify proteins in a complex with PER1 and PER2 at selected circadian phases. The research strategy will make use of mice with tandem epitope-tagged versions of PER1 and PER2 in which the tagged proteins replace the endogenous protein and demonstrably rescue circadian rhythms at the behavioral and molecular level. Liver nuclear and cytoplasmic extracts from control and FH-PER transgenic mice will be subjected to sequential anti-FLAG, anti-Hemagglutinin immunoaffinity purification followed by mass spectrometry. Once a candidate protein has been found to be a constituent of endogenous PER complexes, specific depletion of the endogenous protein by small interfering RNAs (siRNAs) will be used to determine the effect on period length in a validated bioluminescent fibroblast cell line. We expect that the identification of novel constituents of the PERIOD protein complex will yield important clues to clock function and provide new insights into liver physiology. PUBLIC HEALTH RELEVANCE: The overall goal of this proposal is to discover new proteins that are part of the PERIOD protein complex. Identification of these proteins may help elaborate how the circadian clock functions and provide new insights into liver physiology in health and disease.